Cell Lung Cancer Tumors Research Articles

Repeat pulmonary resection for lung malignancies does not affect the postoperative complications: a retrospective study

BackgroundAlthough repeat pulmonary resection (RPR) for multiple lung cancer has been performed for non-small cell lung cancer and metastatic lung tumor, with the prognostic benefit detailed in several reports, the risk of RPR has not been well analyzed.MethodsPatients with lung malignancies who underwent complete resection at Kanazawa Medical University between January 2010 and October 2019 were analyzed. The relationship between postoperative complications and preoperative and perioperative factors was analyzed. Postoperative complications were categorized into five grades according to the Clavien–Dindo classification system.ResultsA total of 41 patients who were received RPR were enrolled in this study. Primary lung tumor was found in 31 patients, and metastatic lung tumor was found in 10 patients. The postoperative complication rate of the first operation was 29%, and that of the second operation was 29%. While there were no significant factors for an increased incidence of postoperative complication in a multivariate analysis, an operation time over 2 h at the second operation tended to affect the incidence of postoperative complication (p = 0.06). Furthermore, the operation time was significantly longer (p = 0.02) and wound length tended to be longer (p = 0.07) in the ipsilateral group than in the contralateral group. The rate of postoperative complications and the length of the postoperative hospital stay were not significantly different between the two groups.ConclusionRPR is safely feasible and is not associated with an increased rate of postoperative complications, even on the ipsilateral side.

Detection of Circulating Tumor Cells in Non-small Cell Lung Cancer and Its Clinical Significance

Detection of Circulating Tumor Cells in Non-small Cell Lung Cancer and Its Clinical Significance

Abstract PO012: Cigarette smoke downregulates mature microRNA-1 in non-small cell cancer (NSCLC) tumor microenvironment

Abstract Rationale: We and others had shown that microRNA-1 (miR-1) is downregulated in the non-small cell lung cancer (NSCLC) tumor microenvironment and that a low mature miR-1 level in the tumor is a predictor of shorter overall survival in lung adenocarcinoma patients. Molecular alterations within the tumor microenvironment can induce cancerization and CS is the most prevalent inducer of NSCLC. In this study we evaluated the clinical associations of miR-1 in clinical samples and its significance as an indicator of cancerization. Methods: We assessed the clinical associations of miR-1 in the tumor (T), adjacent cancer-free (AT), and distant cancer-free (DT) lung tissues of 82 NSCLC patients which were obtained by bronchoscopy and surgery. Ex-vivo cultures of normal human lung tissues and cultured human bronchial epithelial cells, immortalized human endothelial cell were exposed to increasing percentage of CS extract in the growth media and harvested after 24 hours. Mature miR-1 levels and mRNA were measured by Taqman assay and quantitative real time PCR, respectively. Human lung epithelial and endothelial fractions were isolated by Magnetic-activated cell sorting (MACS) technique. For clinical data analysis we used SAS 9.4 proc correction for spearman rank test, proc logistic for regression model analysis and Kaplan-Meier analysis for survival studies. Results: In NSCLC patients, mature miR-1 levels in the tumor had an inverse correlation with the extent of CS exposure (pack-year of smoking). Also, miR-1 levels in AT and DT samples of current smokers were significantly lower than the former smokers, suggesting that CS has a suppressive effect on miR-1 levels. We asked whether CS exposure decreases mature miR-1 levels in the lung tissue. Treatment of ex vivo human lung sample with CS specifically decreased mature miR-1 levels without altering its precursors. This change was observed in both epithelial and endothelial cells isolated from the ex-vivo lungs, as well as cultured cells. Next, we asked whether miR-1 changes in the lungs of NSCLC patients has clinical significance. We found that miR-1 levels in the tumor, AT and DT tissues follows a cancerization gradient. MiR-1 levels were lowest and showed the least variability in the tumors and became higher and more widely distributed in the AT and DT samples. Moreover, mature miR-1 levels in the AT samples had a significant association with overall survival of the patient. Conclusion:Our observations show that CS specifically downregulates mature miR-1 in the NSCLC microenvironment, and together with our previous findings strongly suggests that miR-1 downregulation is an inducer of cancerization. Citation Format: Asawari Korde, Anuradha Ramaswamy, Mark S. Godfrey, Margaret A. Pisani, Jonathan T. Puchalski, Shervin S. Takyar. Cigarette smoke downregulates mature microRNA-1 in non-small cell cancer (NSCLC) tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO012.

Tumor cell proliferation (Ki-67) expression and its prognostic significance in histological subtypes of lung adenocarcinoma

ObjectivesKi-67 is a key molecular marker to indicate the proliferative activity of tumor cells in lung cancer. However, Ki-67 expression and its prognostic significance in histological subtypes of lung adenocarcinoma (LUAD) remain unclear. Materials and MethodsWe retrospectively analyzed 1028 invasive LUAD patients who underwent surgery treatment between January 2012 and April 2020 in our department. Associations between Ki-67 expression and histological subtypes of LUAD, as well as other clinicopathological characteristics, were evaluated. The prognostic role of Ki-67 in LUAD subtypes was further assessed using log-rank test and univariate/multivariate Cox proportional hazards regression analyses. ResultsKi-67 expression differed across LUAD histological subtypes. The solid-predominant adenocarcinoma (SPA, 46.31 ± 24.72) had the highest expression level of Ki-67, followed by micropapillary (MPA, 31.71 ± 18.14), papillary (PPA, 22.09 ± 19.61), acinar (APA, 19.73 ± 18.71) and lepidic-predominant adenocarcinoma (LPA, 9.86 ± 8.10, P < 0.001). Tumors with solid or micropapillary components also had a higher Ki-67 expression than those without solid or micropapillary components. Besides, males, smokers, larger tumor size, lymph node metastasis and EGFR wild type were correlated with elevated Ki-67 expression. Univariate analysis indicated that increased Ki-67 expression and MPA/SPA subtypes were significantly associated with a poorer prognosis. Notably, the survival differences between LUAD subtypes vanished after adjusting for tumor size and Ki-67 expression in multivariate analysis, while Ki-67 was an independent prognostic factor of LUAD. Patients with MPA/SPA had non-inferior overall and disease-free survival than LPA/APA/PPA patients with a Ki-67 expression comparable to MPA/SPA subjects. ConclusionKi-67 expression varied considerably according to the predominant histological subtypes of LUAD. Ki-67 expression level and tumor size contributed to the survival differences between LUAD histological subtypes.

Orchids as Potential Sources of Anticancer Agents: Our Experience

Cancer is the second leading cause of death globally and the cancer burden continues to grow, exerting tremendous physical, emotional and financial strain on individuals, families, communities and health systems. Many health systems in low- and middle-income countries are ill-prepared to manage this burden, and around the world large numbers of cancer patients do not have access to timely quality diagnosis or treatment. Natural products, especially originated from plants, have been applied as remedies to treat various diseases, including cancer, for thousands of years. Several anticancer agents derived from plants, such as paclitaxel (taxol), vinblastine, vincristine, camptothecin derivatives, podophyllotoxins, are in clinical use and a number of other promising agents are in preclinical or clinical development. A growing number of bioactive compounds with cytostatic and cytotoxic activities to treat cancer have been isolated from several orchid species. The Annapurna Research Center in collaboration with other academic institutions is engaged in identifying and exploring anticancer compounds in orchids of Nepal. The center has identified ten different orchid species which contain high phenol and flavonoid contents evidently showing anticancer activities in our in vitro assays against different cancer cell lines viz cervical cancer, lung cancer and brain tumor cell lines. These findings demonstrate that orchids which have been used in in various formulations in traditional medicine since ancient times qualify as potential source for novel drug candidates or starting points for further lead optimization towards clinical candidates for the most urgently needed treatment of aggressive types of cancer. This review paper highlights that in all likelihood; our contributions in orchid research here in Nepal could bring great relief to patients with cancer globally.

DNA methylome profiling of circulating tumor cells in lung cancer at single base-pair resolution

DNA methylation plays a pivotal role in regulating cellular processes, and altered DNA methylation pattern is a general hallmark of cancer. However, DNA methylome in circulating tumor cells (CTCs) is still a mystery due to the lack of proper analytical techniques. We introduced an efficient workflow, LCM–µWGBS, which can efficiently profile the DNA methylation of microdissected CTC samples. LCM–µWGBS combines the laser capture microdissection (LCM)-based CTC capture method and whole-genome bisulfite sequencing in very small CTC population (µWGBS) to gain insight into the DNA methylation landscape of CTCs. We herein profiled the DNA methylome of CTCs from lung cancer patients. Deriving from a comprehensive analysis of CTC methylome, a unique “CTC DNA methylation signature” that is distinct from primary lung cancer tissues was identified. Further analysis showed that promoter hypermethylation of epithelial genes is a hallmark of stable epithelial–mesenchymal transition process. Moreover, it has been suggested that CTCs are endowed with a stemness-related feature during dissemination and metastasis. This work constitutes a unique DNA methylation analysis of CTCs at single base-pair resolution, which might facilitate to propose noninvasive CTC DNA methylation biomarkers contributing to clinical diagnosis.

Preparation of Programmed Cell Death-Ligand 1 Antibody Nanoparticles Based on Nude Mouse Model and Its Therapeutic Effect on Lung Cancer.

In this experiment, the advantages of both liposomes and polymer nanoparticles were used to synthesize adriamycin-loaded liposome-polymer nanoparticles and conjugate them with PD-L 1 antibody (referred to as PD-L 1 antibody nano particles). The nanoparticles were encapsulated with doxorubicin hydrochloride and the near-infrared dye DIR, and small animal imaging methods were used in animal experiments to evaluate the targeting and therapeutic effects. The results showed that the red fluorescence of doxorubicin hydrochloride entered the cells, and the red fluorescence of the PD-L1 antibody nanoparticles in the 4h group was better than that in the 2h group. The intracellular red fluorescence of PD-L1 antibody nanoparticles 4h group was stronger than that of free doxorubicin 4h group. Flow cytometry and confocal experiments showed that A549 cells took up more PD-L1 antibody nanoparticles. The results showed that the fluorescence intensity of the PD-L1 antibody nanoparticle group was significantly stronger than that of the nanoparticle group, and the tumor outline was clear, and the fluorescence intensity became stronger and stronger over time, indicating that the PD-L1 antibody nanoparticles were targeted. Has certain targeting capabilities. The PD-L1 antibody nanoparticles synthesized in this study are a good drug carrier targeting lung cancer tumor cells, which can be better taken up by A549 lung cancer cells, and can more effectively kill tumor cells, inhibit tumor growth, and wrap near-infrared dyes are more conducive to in vivo imaging of animals and are useful for observing the effects of targeted treatment of lung cancer.

IL-9 Producing Tumor-Infiltrating Lymphocytes (Til) and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer

Although lung cancer is the leading cause of cancer death worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro , IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of lung pSTAT3, Th17 and Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production and induction of IL-21 in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer. Funding: This work was supported by DFG grant FI817-5-1,2 and by a DFG FI817- 5-3 granted to SF. M.T.C. work is supported by a DFG CH 1428/2-1. MHK was supported by NIH grant AI057459. Declaration of Interest: None to declare. Ethical Approval: Our human study was performed at the Friedrich-Alexander-University Erlangen-Nurnberg, Germany, after being approved by the ethics review board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: DRK5376).

Design and Biological Evaluation of 3-Aryl-4-alkylpyrazol-5-amines Based on the Target Fishing.

Pyrazol-5-amine derivatives are an important class of heterocyclic compounds. However, there are less 4-alkyl substituted pyrazoles reported. Here reported are the design, synthesis and biological evaluation of 3-aryl-4- alkylpyrazol-5-amines derivatives. A serials of 3-aryl-4-alkylpyrazol-5-amines were designed and the biological action targets were screened by target fishing function of Discovery Studio software. The synthesis route involved 3-oxo-3-arylpropanenitrile formation, alkylation, pyrazole formation, and amides formation. The antitumor activities of these compounds were carried out by thiazolyl blue tetrazolium bromide (MTT) method using U-2 OS (osteosarcoma) and A549 (lung cancer) tumor cells. Eight 3-aryl-4-alkylpyrazol-5-amines were synthesized, and their structures were verified by 1H NMR, 13C NMR, and HRMS. Thirteen pharmacophores were mapped out by target fishing. Compound 5h showed anti-proliferation activities against U-2 OS and A549 tumor cell with IC50 value of 0.9 μM and 1.2 μM, respectively. Compound 5h might represent a promising scaffold for the further development of novel antitumor drugs.

Prolonged immune checkpoint inhibitor response of a patient with a RET rearranged non-small cell lung cancer and high tumour mutational burden: case report and review of the literature

Prolonged immune checkpoint inhibitor response of a patient with a RET rearranged non-small cell lung cancer and high tumour mutational burden: case report and review of the literature

Prolonged immune checkpoint inhibitor response of a patient with a RET rearranged non-small cell lung cancer and high tumour mutational burden: case report and review of the literature

Prolonged immune checkpoint inhibitor response of a patient with a RET rearranged non-small cell lung cancer and high tumour mutational burden: case report and review of the literature

Abstract 5368: A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer

Abstract Lung cancer biopsy can be difficult to perform successfully; it has a 30% adverse event risk and often provides insufficient material to test. Therefore, a liquid biopsy method to analyze the tumor is advantageous. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression. HER2 is a well-known therapeutic target in breast cancer and studies have shown that HER2 status on CTCs may provide prognostic information about response to anti-HER2 therapies. Less is understood about its frequency and clinical importance in non-small cell lung cancer (NSCLC). The RareCyte platform is uniquely suited for CTC identification and phenotypic characterization with a sensitive, accurate, simple and repeatable workflow from blood collection to CTC characterization and single-cell isolation. We developed an assay for detecting HER2 expression on lung CTCs using the RareCyte platform and validated the assay on model CTC spike-in samples with high (BT-474), medium (MDA-MB-453), low (H1650 and OVCAR-3) and absent (MDA-MB-468) HER2 protein levels. Blood was processed to slides using the AccuCyte Sample Preparation System and stained with anti-HER2 together with the RarePlex Kit for CTC identification (nuclear dye, anti-CD45 to exclude WBC, and anti-cytokeratin/EpCAM for CTCs). The spike-in samples showed the expected trend in per-cell HER2 mean fluorescence intensity values. The percentage of HER2-positive cells was &amp;gt; 90% for the HER2-high, medium and low cell lines, and &amp;lt; 5% for the HER2-negative cell line. The assay was applied to 10 advanced stage (III or IV) post-treatment NSCLC patient samples. At least 1 CTC was found in 6 samples (range 0-2043) and HER2 expression was confirmed in 5 out of the 6 samples, indicating this assay is useful for characterizing HER2 expression in CTCs in NSCLC. Citation Format: Edward Lo, Daniel Campton, Lillian Costandy, Heather Itamoto, Ryan Huston, Braden Gardner, Steve Reese, Arturo Ramirez, Eric P. Kaldjian, Tad George, I-Ming Wang, Steven Pirie-Shepherd. A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5368.

Abstract 775: Longitudinal investigation of PD-L1 expression on circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab

Abstract Purpose: We have previously reported that PD-L1-positive rate on CTCs at baseline was correlated with response to nivolumab in NSCLC patients. Here, we conducted a longitudinal follow-up investigation of PD-L1 expression on CTCs and its association with clinical outcome. Methods: For CTC detection, 3ml of peripheral blood was collected from advanced NSCLC patients treated with nivolumab at baseline and week 4, 8, 12 and 24 or until progressive disease (PD). CTCs were enriched by microcavity array system based on the difference in size and defined as DAPI-positive, CK-positive, and CD45-negative cells and evaluated for PD-L1 expression by additional immunostaining. Results: Forty-five patients were enrolled between January 2016 and January 2018 at Wakayama Medical University. The patient characteristics were as follows: median age 68 years (range, 49-86); male 75%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/ other, 68/25/7%; partial response/stable disease/PD/not evaluable, 20/25/45/9%. At baseline, CTCs were evaluated in 44 patients (median, 13; range, 1-104) and PD-L1 positive CTCs were detected in 82% patients (median 29%; range, 0-100%), at week 4, evaluated in 31 patients (median, 4; range, 0-115) and detected in 58% patients (median, 13%; range, 0-100%), at week 8, evaluated in 16 patients (median, 11; range, 1-276) and detected in 56% patients (median, 7%; range, 0-60%), at week 12, evaluated in 13 patients (median, 11; range, 0-449) and detected in 62% patients (median, 15%; range, 0-88%), and at week 24, evaluated in 11 patients (median, 8; range, 0-35) and detected in 55% patients (median, 26%; range, 0-92%). Significant decrease in the ratio of PD-L1 positive CTCs between week 4 to week 8 was determined by Wilcoxon-matched-pairs signed rank test. Cutoff value of PD-L1-positivity rates in CTCs to segregate durable clinical benefit (DCB) from non-DCB was calculated to be 7% at week 8 according to ROC curve. Progression free survival was significantly longer in the patients with 7% or more of PD-L1 positivity rates (n = 8) than in those with less than 7% (n = 8) (p &amp;lt; 0.01) at week 8, suggesting the predictive significance of early evaluation of PD-L1 expression on CTCs after nivolumab treatment. Interestingly, CTC count in 7 out of 9 patients who had a partial response (PR) transiently increased from the one immediately prior to achieving a PR. Progression free survival was significantly longer in patients with 1.5 times or more increase of CTC count (n = 6) than in those with less than 1.5 times (n = 3) (p &amp;lt; 0.05), suggesting the transient tumor cell intravasation due to nivolumab treatment and its potential correlation with the duration of response depending on its magnitude. Conclusions: Longitudinal evaluation of PD-L1-expressing CTCs may have a predictive potential in NSCLC patients treated with nivolumab. Citation Format: Yasuhiro Koh, Mio Ikeda, Shunsuke Teraoka, Koichi Sato, Nahomi Tokudome, Atsushi Hayata, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Longitudinal investigation of PD-L1 expression on circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 775.

Abstract 4989: Introduction of a robust workflow for the whole-side acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay

Abstract Background: Profiling multiple cell types in tissue samples requires more markers than can be supported by conventional approaches to immunohistochemistry. InSituPlex® multiplex immunofluorescence (mIF) technology enables biomarker multiplexing in tissue samples with an automated workflow to support discovery of immune cell signatures in the tumor immune microenvironment (TiME). Here we have designed and tested an 8-plex immunophenotyping panel using the InSituPlex approach to detect and classify T cells, macrophages, and tumor cells in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) FFPE tissue. Methods: An 8-plex UltiMapper™ immunofluorescence was developed to stain FFPE tissue, including colorectal cancer, human tonsil, and non-small cell lung cancer. Each sample was stained for the following 8 markers: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, and pan-CK. Whole-slide imaging was performed in two imaging rounds on the RareCyte CyteFinder II HT slide scanner, and same-slide image alignment and phenotypic analysis was performed using the HALO Highplex FL module from Indica Labs. Results: Co-registration of two 4-plex imaging rounds and fusing into a single image file allowed for the seamless cell phenotyping of markers across imaging rounds. The 8-plex assay and workflow is compatible across multiple tumor sample types. The samples were characterized based on binned expression and co-expression of biomarkers. The following major cell phenotypes were detected: PD-L1 checkpoint expression, T cells (CD3+), cytotoxic T cells (CD3+/CD8+), T-helper cells (CD3+/CD4+), regulatory T-cells (CD3+/CD4+/FoxP3+), exhausted T cells (CD3+/PD-1+), macrophages (CD68+), and tumor cells (pan-CK+). Conclusions: The 8-plex assay workflow involving InSituPlex mIF, CyteFinder HT scanner, and HALO analysis supports the ability to measure biomarkers in situ in streamlined and scalable approach. Citation Format: Anne E. Hellebust, Kyla Teplitz, Katir K. Patel, Karan Sharma, Amanda Bares, Mael Manesse, Mark Burton, Bonnie Phillips, Sean Downing, Kate Lillard. Introduction of a robust workflow for the whole-side acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4989.

Abstract 2485: RNA editing by APOBEC3s in lung cancer tumors largely occurs in infiltrating immune cells

Abstract Introduction: APOBEC3 enzymes contribute significantly to DNA mutagenesis in cancer. Our recent studies show that these enzymes are also capable of converting C bases at specific positions (sites) of RNAs to U. By examining primary non-small cell lung cancer (NSCLC) tumors of The Cancer Genome Atlas (TCGA) project, we have demonstrated that such APOBEC3-mediated C-to-U RNA editing occurs in lung cancer tumors. While we could detect editing events in the tumors, their cellular origin remains unclear. Here, we investigated if the editing occurs in cancerous or stromal cells of lung cancer tumors. Methods: Immune-related features of the TCGA lung cancer tumors (n = 1,009) were obtained from various TCGA studies to examine their association with APOBEC3-mediated RNA editing that we had determined previously. Freshly resected late-stage NSCLC tumors of five patients were mechanically and enzymatically dissociated into single cells using a cocktail of collagenase, elastase, and DNAse enzymes. Antibodies conjugated to magnetic beads were then used to isolate cancerous epithelial (EpCAM+) and stromal pan-immune (EpCAM-/CD45+) and macrophage/monocyte (EpCAM-/CD14+) subsets of cells from the single-cell suspensions. RNA (depleted of rRNA) and genomic DNA of the cell isolations were subjected to total RNA and whole exome sequencing. Sequencing data was examined to identify C-to-U RNA editing at 5,208 known APOBEC3-mediated editing sites. Results: TCGA lung tumors with higher RNA editing levels had stronger tumoral immune response. Particularly, they had about 25% more infiltrating leukocytes, CD8 T cell fraction, and immune cytolytic activity compared to tumors with low editing level (P &amp;lt; 0.01 in all t tests). As expected, expression of genes that are markers of epithelial (such as EPCAM and MUC1), pan-immune (CD45, ITGAL), and macrophage/monocyte (CD14, FCGR3A) cells were dramatically higher among the corresponding cell subsets that were isolated from lung tumors. Whereas expression of APOBEC3B and F were similar among the three subsets, the expression of APOBEC3A, C, D, G and H were 2-4-fold higher in pan-immune and macrophage/monocyte compared to epithelial cells. Concordantly, while editing events were seen in the epithelial cells at average 7% of examined sites, editing was more frequent in the pan-immune and macrophage/monocyte cells (17% and 22%, respectively). Conclusions: RNA editing by APOBEC3s reflects a heightened immune response in lung cancer. A large proportion of RNA editing by APOBEC3s in tumors occurs in immune cells such as macrophages. Further investigations are needed to understand RNA editing in various other immune cells, as well as the relevance of RNA editing of specific genes in cancer and immune cell biology. Citation Format: Mariko Asaoka, Eric Kannisto, Takashi Ishikawa, Sai Yendamuri, Santosh Kumar Patnaik. RNA editing by APOBEC3s in lung cancer tumors largely occurs in infiltrating immune cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2485.

Microsieves for the detection of circulating tumor cells in leukapheresis product in non-small cell lung cancer patients.

BackgroundCirculating tumor cells (CTC) in non-small cell lung cancer (NSCLC) patients are a prognostic and possible therapeutic marker, but have a low frequency of appearance. Diagnostic leukapheresis (DLA) concentrates CTC and mononuclear cells from the blood. We evaluated a protocol using two VyCAP microsieves to filter DLA product of NSCLC patients and enumerate CTC, compared with CellSearch as a gold standard.MethodsDLA was performed in NSCLC patients before starting treatment. DLA product equaling 2×108 leukocytes was diluted to 9 mL with CellSearch dilution buffer in a Transfix CTC tube. Within 72 hours the sample was filtered with a 7 µm pore microsieve and subsequently over a 5µm pore microsieve. CTC were defined as nucleated cells which stained for cytokeratin, but lacked CD45 and CD16. CellSearch detected CTC in the same volume of DLA.ResultsOf 29 patients a median of 1.4 mL DLA product (range, 0.5–4.1) was filtered (2% of total product) successfully in 93% and 45% of patients using 7 and 5 µm pores, respectively. Two DLA products were unevaluable for CTC detection. Clogging of the 5 µm but not 7 µm microsieves was positively correlated with fixation time (ρ=0.51, P<0.01). VyCAP detected CTC in 44% (12/27) of DLA products. Median CTC count per mL DLA was 0 [interquartile range (IQR): 0–1]. CellSearch detected CTC in 63% of DLA products (median =0.9 CTC per mL DLA, IQR: 0–2.1). CTC counts detected by CellSearch were significantly higher compared with VyCAP (P=0.05).ConclusionsVyCAP microsieves can identify CTC in DLA product, but workflows need to be optimized.

Xanthohumol targets the ERK1/2‑Fra1 signaling axis to reduce cyclin D1 expression and inhibit non‑small cell lung cancer.

High expression of cyclin D1 has a crucial role in the maintenance of unlimited cell growth in human cancer cells. The present study indicated that cyclin D1 was overexpressed in human non-small cell lung cancer (NSCLC) tumor tissues and cell lines. Knockout of cyclin D1 suppressed NSCLC cell growth, colony formation and in vivo tumor growth. Of note, the natural product xanthohumol (Xanth) inhibited NSCLC cells via the downregulation of cyclin D1. A further mechanistic study revealed that Xanth suppressed ERK1/2 signaling and reduced the protein levels of FOS-related antigen 1 (Fra1), which eventually inhibited the transcriptional activity of activator protein-1 and decreased the mRNA level of cyclin D1. Furthermore, suppression of ERK1/2 impaired Fra1 phosphorylation and enhanced Xanth-induced Fra1 ubiquitination and degradation. In addition, the S265D mutation compromised Xanth-induced Fra1 degradation. Finally, the in vivo anti-tumor effect of Xanth was validated in a xenograft mouse model. In summary, the present results indicated that targeting ERK1/2-Fra1-cyclin D1 signaling is a promising anti-tumor strategy for NSCLC treatment.

Over-expression of long non-coding RNA ZEB2-AS1 may predict poor prognosis and promote the migration, invasion, and epithelial-mesenchymal transition of tumor cells in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains the most common cause of human cancer-related death worldwide, and the present study aims to explore the roles of long non-coding (lnc)RNA ZEB2-AS1 in NSCLC and the related mechanism. Quantitative real-time-polymerase chain reaction was performed to compare the expressions of ZEB2-AS1 in NSCLC cancer tissue and the adjacent non-tumorous tissues. The diagnostic and prognostic roles of ZEB2-AS1 in NSCLC were also evaluated by the receiver operating characteristic curve and the Kaplan-Meier survival analysis. NSCLC cell line A549 cells were transfected with ZEB2-AS1 siRNA, and the cell viability, migration, invasion, and epithelial-mesenchymal transition (EMT) of the ZEB2-AS1 siRNA group and control group were compared. ZEB2-AS1 was significantly increased in NSCLC tissues. The knockdown of ZEB2-AS1 markedly inhibited the cell viability, migration, invasion, and EMT of A549 cells in vitro. ZEB2-AS1 was up-regulated in NSCLC, and it may serve as a potential target for the diagnosis, prognosis, and treatment of NSCLC.

Radiomics study for predicting the expression of PD-L1 in non-small cell lung cancer based on CT images and clinicopathologic features.

To predict programmed death-ligand 1 (PD-L1) expression of tumor cells in non-small cell lung cancer (NSCLC) patients by using a radiomics study based on CT images and clinicopathologic features. A total of 390 confirmed NSCLC patients who performed chest CT scan and immunohistochemistry (IHC) examination of PD-L1 of lung tumors with clinic data were collected in this retrospective study, which were divided into two cohorts namely, training (n = 260) and validation (n = 130) cohort. Clinicopathologic features were compared between two cohorts. Lung tumors were segmented by using ITK-snap kit on CT images. Total 200 radiomic features in the segmented images were calculated using in-house texture analysis software, then filtered and minimized by least absolute shrinkage and selection operator (LASSO) regression to select optimal radiomic features based on its relevance of PD-L1 expression status in IHC results and develop radiomics signature. Radiomics signature and clinicopathologic risk factors were incorporated to develop prediction model by using multivariable logistic regression analysis. The receiver operating characteristic (ROC) curves were generated and the areas under the curves (AUC) were reckoned to predict PD-L1 expression in both training and validation cohorts. In 200 extracted radiomic features, 9 were selected to develop radiomics signature. In univariate analysis, PD-L1 expression of lung tumors was significantly correlated with radiomics signature, histologic type, and histologic grade (p < 0.05, respectively). However, PD-L1 expression was not correlated with gender, age, tumor location, CEA level, TNM stage, and smoking (p > 0.05). For prediction of PD-L1 expression, the prediction model that combines radiomics signature and clinicopathologic features resulted in AUCs of 0.829 and 0.848 in the training and validation cohort, respectively. The prediction model that incorporates the radiomics signature and clinical risk factors has potential to facilitate the individualized prediction of PD-L1 expression in NSCLC patients and identify patients who can benefit from anti-PD-L1 immunotherapy.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA19.